Humans, like all organisms, are subject to environmental stresses. To deal with these stresses, such as damage to the DMA or oxidation of proteins and lipids, our cells possess numerous protective mechanisms (e.g. DMA repair or anti-oxidant systems). However, when stresses become too great, our cells are programmed to kill themselves-a process called programmed cell death or apoptosis. Programmed cell death plays important roles in human health and disease;excessive programmed cell death leads to the significant morbidity and mortality associated with ischemic heart disease and stroke, whereas defective cell death leads to the second greatest cause of mortality in the United States, cancer. Sphingolipids are important regulators of cell growth and death. Ceramide, a bioactive sphingolipid, is an essential mediator of programmed cell death. Ceramide is produced by ceramide synthases (CerSes) during cell death, and there is an accumulating body of evidence that these enzymes play specific roles in regulating ceramide generation and cell death. However, the mechanisms of CerS- and ceramide-mediated cell death are ill-defined and warrant further study. By understanding the details of these pathways we will be able to identify targets of therapeutic interest. The goal of this project is to determine the role of CerSS and CerS6 in mediating programmed cell death. This project will utilize the MCF-7 breast adenocarcinoma cell line to investigate the contribution of CerSS and CerS6 to cell death induced by the genotoxic stress ultraviolet light (UVC). In preliminary investigations, the pro-death protein Bax has emerged as a downstream target of CerSS and CerS6- mediated ceramide production in UVC-induced death. The aims of this study are 1) determine how CerSS and CerS6 regulate cell death;2) determine how CerSS and CerS6 are regulated during cell death;and 3) determine whether CerSS or CerS6-mediated ceramide production is sufficient to activate cell death pathways (e.g Bax). The primary methodology of these studies will be to overexpress or knockdown CerSS and CerS6 and examine subsequent effects on Bax activation and other parameters of cell death. This project represents an incremental step in achieving our long-term goal of determining the mechanisms of ceramide-mediated programmed cell death. Human cells respond to excessive stress by making a decision to kill themselves. This project seeks to determine how the lipid ceramide and the enzymes that make ceramide regulate this decision.